ABSTRACT Little is known about cellular and extracellular composition of fibrosis in bone marrows in the context of human immunodeficiency virus/acquired immunodeficiency syndrome.
To evaluate the stromal composition of bone marrows affected by human immunodeficiency virus/ acquired immunodeficiency syndrome and to correlate this with laboratory parameters including CD4 lymphocyte counts.
We evaluated extracellular matrix and stromal cell composition in bone marrows and correlated these results with hematologic parameters. Extracellular matrix, stromal cells, and smooth muscle differentiation were evaluated by immunohistochemistry for collagen type IV expression and reticulin staining, an antibody directed against low-affinity nerve growth factor receptor (a marker of adventitial reticular cells), and actin staining, respectively. Concurrent laboratory information was collected, including white blood cell count, hemoglobin, platelet count, CD4 count, CD8 count, CD4/CD8 ratio, and absolute lymphocyte count.
Bone marrows of 35 patients with human immunodeficiency virus/acquired immunodeficiency syndrome were evaluated.
Correlation of reticulin, low-affinity nerve growth factor receptor, actin, and collagen IV staining with hematologic parameters.
More than half of the bone marrows showed moderate to severe reticulin fibrosis. The degree of reticulin fibrosis was correlated with the degree of low-affinity nerve growth factor receptor expression (P = .048). Actin expression was identified in only 3 of 35 cases and collagen IV in only 5 of 35 cases. No statistical relationship between degree of fibrosis and CD4 count was identified. Lower levels of low-affinity nerve growth factor receptor expression were associated with CD4 counts of >100 (P = .04). Marrow fibrosis was present in almost all cases studied (97%), and the staining of adventitial reticular cells correlated with the degree of reticulin fibrosis.
There does not appear to be a correlation between CD4 count and degree of fibrosis, suggesting that the mechanism of fibrosis is independent of disease status.

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Arch Pathol Lab Med—Vol 129, September 2005Marrow Stroma in HIV/AIDS—O’Malley et al1137Evaluation of Stroma in Human ImmunodeficiencyVirus/Acquired Immunodeficiency Syndrome–AffectedBone Marrows and Correlation With CD4 CountsDennis P. O’Malley, MD; Joy Sen, MD; Beth E. Juliar, MA, MS; Attilio Orazi, MD, FRCPath● Context.—Little is known about cellular and extracellularcomposition of fibrosis in bone marrows in the context ofhuman immunodeficiency virus/acquired immunodeficien-cy syndrome.Objective.—To evaluate the stromal composition ofbone marrows affected by human immunodeficiency virus/acquired immunodeficiency syndrome and to correlate thiswith laboratory parameters including CD4 lymphocytecounts.Design.—We evaluated extracellular matrix and stromalcell composition in bone marrows and correlated these re-sults with hematologic parameters. Extracellular matrix,stromal cells, and smooth muscle differentiation were eval-uated by immunohistochemistry for collagen type IV ex-pression and reticulin staining, an antibody directedagainst low-affinity nerve growth factor receptor (a markerof adventitial reticular cells), and actin staining, respec-tively. Concurrent laboratory information was collected,including white blood cell count, hemoglobin, plateletcount, CD4 count, CD8 count, CD4/CD8 ratio, and ab-solute lymphocyte count.Patients.—Bone marrows of 35 patients with human im-munodeficiency virus/acquired immunodeficiency syn-drome were evaluated.Main Outcome Measures.—Correlation of reticulin,low-affinity nerve growth factor receptor, actin, and collagenIV staining with hematologic parameters.Results.—More than half of the bone marrows showedmoderate to severe reticulin fibrosis. The degree of retic-ulin fibrosis was correlated with the degree of low-affinitynerve growth factor receptor expression (P ? .048). Actinexpression was identified in only 3 of 35 cases and collagenIV in only 5 of 35 cases. No statistical relationship betweendegree of fibrosis and CD4 count was identified. Lowerlevels of low-affinity nerve growth factor receptor expres-sion were associated with CD4 counts of ?100 (P ? .04).Marrow fibrosis was present in almost all cases studied(97%), and the staining of adventitial reticular cells cor-related with the degree of reticulin fibrosis.Conclusions.—There does not appear to be a correlationbetween CD4 count and degree of fibrosis, suggesting thatthe mechanism of fibrosis is independent of disease status.(Arch Pathol Lab Med. 2005;129:1137–1140)Tdrome (AIDS) can be challenging. The constellation ofchanges, termed HIV myelopathy, includes a wide range ofabnormal findings and can mimic a variety of primarybone marrow disorders, including myelodysplastic syn-dromes and myeloproliferative disorders.1In addition,HIV/AIDS is associated with an increased incidence ofopportunistic infections, hematologic and nonhematologiche bone marrow pathology of human immunodeficien-cy virus (HIV)/acquired immunodeficiency syn-Accepted for publication May 12, 2005.From the Department of Pathology and Laboratory Medicine, Divi-sion of Hematopathology (Drs O’Malley and Orazi), the Departmentof Pathology and Laboratory Medicine (Dr Sen), and the Departmentof Medicine, Division of Biostatistics (Ms Juliar), Indiana UniversitySchool of Medicine, Indianapolis.The authors have no relevant financial interest in the products orcompanies described in this article.Presented in abstract form at the annual meeting of the United Statesand Canadian International Academy of Pathology, Vancouver, BritishColumbia, March 2004.Reprints: Dennis P. O’Malley, MD, Indiana University School ofMedicine, Department of Pathology and Laboratory Medicine, Divisionof Hematopathology, 702 Barnhill Dr, Riley 0969, Indianapolis, IN46202 (e-mail: dpomalle@iupui.edu).malignancies, and other hematologic complications, whichtypically worsen as the disease progresses.2Bone marrow stroma is complex; it is a heterogeneousmixture of cellular and extracellular components that con-tribute to the unique microenvironment of the marrowcavity. Bone marrow stromal responses are a commonpathway in both benign and malignant marrow disordersand account for some of the most striking changes seenin HIV/AIDS bone marrow. Defects of the bone marrowmicroenvironment contribute to the development of he-matologic abnormalities in HIV/AIDS.2,3It has beenshown that stromal elements are directly infected and mayact as a reservoir for the HIV virus.2–4The HIV virus di-rectly infects a variety of stromal elements, includingmonocytic/macrophage elements, microvascular endothe-lial cells, and fibroblastic and myoid cells.2–5Infection ofthe stromal elements leads to a loss of the hematopoieticsupport function.6In addition, megakaryocytes, whichplay an active role in the makeup of bone marrow stroma,are also infected by the HIV virus, as demonstrated by thepresence of CD4 receptors on their surface.7Human im-munodeficiency virus infection of bone marrow elementsmay lead to loss of hematopoietic support function and

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1138Arch Pathol Lab Med—Vol 129, September 2005Marrow Stroma in HIV/AIDS—O’Malley et alTable 1. Antibodies Used to Characterize BoneMarrow Stroma*Antibody (Clone)DilutionMain SpecificityLNGFR (ME20-4) 1:80ARC, DRC, stroma cellsubsetsBasement membrane-typecollagenMyofibroblasts, smoothmuscle cellsCollagen IV (CIV22)1:100Actin HHF35 (1A4)1:100* LNGFR indicates low-affinity nerve growth factor receptor; ARC,adventitial reticular cells; and DRC, dendritic reticulum cells.may be responsible for some of the hematologic abnor-malities.3These changes may ultimately lead to cytopeniasand susceptibility to infections, which are significantprob-lems in the HIV-positive population.We attempted to characterize the cellular and extracel-lular stromal reaction seen in the bone marrow in HIV/AIDS, using an approach similar to that used to evaluatestromal changes in fibrotic myeloproliferative disordersand metastatic malignancy.8The extracellular matrix was evaluated by reticulinstaining and by immunohistochemistry for collagen typeIV expression. Both reticulin fibers and collagen IV areknown to be markedly increased in fibrotic marrow states,such as chronic idiopathic myelofibrosis.8–10Low-affinitynerve growth factor receptor (LNGFR) has been recentlydescribed as a specific marker for a subset of bone marrowstromal cells known as adventitial reticular cells (ARC).11,12In previous studies, the intensity of bone marrow LNGFRstaining has been found to correlate with the degree ofreticulin fibrosis.8–13Actin staining was used to identifymyofibroblastic differentiation in bone marrow stromalcells.14Recent evidence has shown that bone marrow my-oid cells may represent marrow reticular cells undergoingcytoskeletal remodeling in response to various stimuli,such as the presence of metastatic carcinoma.15Because CD4?T-lymphocyte counts are regularly usedas a marker of disease progress and response to therapyin HIV/AIDS, we attempted to test correlation of the ob-served stromal responses with CD4?lymphocyte countsand other hematologic parameters.MATERIALS AND METHODSBone marrow biopsies of patients with HIV/AIDS were ob-tained from the files of Wishard Memorial Hospital and ClarianHealth Partners (Indianapolis, Ind). In all, formalin-fixed, paraf-fin-embedded bone marrow biopsies of 35 patients were studied.In all cases, either significant hematologic abnormalities or pos-sible involvement by infectious disease prompted bone marrowexamination. Concurrent clinical information was collected in-cluding age, white blood cell count, hemoglobin, platelet count,CD4 count, CD8 count, CD4/CD8 ratio, and absolute lymphocytecount. There was no evidence on chart review of transfusionswithin 7 days preceding the bone marrow biopsies.Standard hematoxylin-eosin sections were evaluated in all cas-es. Bone marrow stroma was evaluated using immunohistochem-ical staining for collagen type IV (CIV; DakoCorporation, Car-pinteria, Calif), actin (HHF-35; Dako), and LNGFR (ME20-4;Dako) expression (Table 1) and histochemical staining for retic-ulin. Density of stromal cell staining was graded semiquantita-tively, as follows: 0? indicates absent or rare; 1?, focal weakstaining; 2?, focally intense or diffuse staining; and 3?, diffuseintense staining.Immunoperoxidase stains were performed using an automatedimmunostainer (Dako), which employs a standard streptavidin-biotin-peroxidase complex technique. Endogenous peroxidase ac-tivity was blocked with 3% hydrogen peroxide in methanol, andendogenous biotin activity was blocked using avidin and biotin.The peroxidase activity was developed with 3,3-diaminobenzi-dine and counterstained with hematoxylin. Controls were eval-uated and stained appropriately in all cases.Extracellular MatrixThe extracellular matrix was evaluated by reticulin staining,using the Gomori technique, and by immunohistochemistry forcollagen type IV expression.Stromal Cell CompositionLow-affinity nerve growth factor receptor staining was used toidentify bone marrow stromal ARCs. Actin staining was used toevaluate myofibroblastic differentiation in bone marrow stromalcells.14Statistical AnalysisStatistical analyses were performed using LogXact (version 2.1)(Cytel Software Corporation, Cambridge, Mass) and SAS (version8.2) (SAS Institute Inc, Cary, NC) software packages using a P of.05 for all tests performed. Comparison of CD4 (?100 or ?100)and LNGFR/reticulin staining was performed using exact logis-tic regression. Comparison of CD4 count as a continuous variableand LNGFR/reticulin staining was performed using linear re-gression. For analyses of CD4 count, LNGFR and reticulin stain-ing were tested separately in univariate and together in multipleregressions. The association between LNGFR and reticulin stain-ing was analyzed using the Cochran-Mantel-Haenszel test.RESULTSDemographics and Hematologic ParametersThe patients included in the study consisted of 9 womenand 26 men. The patients’ ages ranged from 18 to 57 years,with a mean age of 38 years. The mean hematologic pa-rameters were as follows: white blood cell count, 4.3 ?103/?L; hemoglobin, 10.6 g/dL; and platelet count, 140 ?103/?L. The mean absolute lymphocyte count was 1014/?L (normal range, 1400–3800/?L), with all but 1 patienthaving a decreased CD4/CD8 ratio (?0.9) and a meanCD4/CD8 ratio of 0.3. The CD4?lymphocyte count hada wide range (1–928/?L) (normal range, 550–1600/?L),with 21 of 35 patients having an absolute CD4 count lessthan 100. We chose to compare patients with CD4?lym-phocyte counts of less than 100 CD4?cells/?L with pa-tients with 100 or more CD4?cells/?L. The CD8 countsalso varied widely (19–1961/?L) (normal range, 200–700/?L), with an average of 572/?L. The results are summa-rized in Table 2.Histologic ParametersHematoxylin-eosin–stained sections werereviewed in all cases (Figure, A).Reticulin Fibrosis and LNGFR.all cases showed at least 1? reticulin fibrosis (0?, 1 case;1?, 15 cases; 2?, 15 cases; and 3?, 4 cases). A major pro-portion of cases (19/35, or 54%) showed moderate or se-vere reticulin fibrosis (Figure, B). All cases but one showedan increase in LNGFR staining (0?, 1 case; 1?, 15 cases;2?, 15 cases; and 3?, 4 cases) (Figure, C). In cases withincreased reticulin fibrosis, the expression of LNGFR inARC was typically upregulated. The degree of reticulinfibrosis paralleled the degree of LNGFR expression (P ?.048).Actin and Collagen IV .was identified focally in only 9% (3/35) of cases (Figure,Histology.With one exception,Actin (HHF-35) expression

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Arch Pathol Lab Med—Vol 129, September 2005Marrow Stroma in HIV/AIDS—O’Malley et al1139Table 2. Summary of Results*?100 CD4?Cells/?L(n ? 14)?100 CD4?Cells/?L(n ? 21)Age, yWBC, ?103/?LHemoglobin, g/dLPlatelets, ?103/?LALC, /?LCD4, /?LCD8, /?LCD4/CD8 ratioActin†LNGFR†Reticulin†Collagen IV†* WBC indicates white blood cell; ALC, absolute lymphocyte count;and LNGFR, low-affinity nerve growth factor receptor.† Mean values of graded scores on a scale of 0? to 3?, as follows:0? indicates absent or rare; 1?, focal weak staining; 2?, focally in-tense or diffuse staining; and 3?, diffuse intense staining.354.911.91601779329773403.89.6127724354390.10.051.861.670.190.50.141.291.570.07A, Human immunodeficiency virus/acquired immunodeficiency syndrome bone marrow. Hypercellular bone marrow with increased megakaryo-cytes and plasma cells mimicking a myeloproliferative disorder (hematoxylin-eosin, oil immersion, original magnification ?500). B, Reticulinstaining. Increased number and diffuse distribution of reticulin fibrils corresponding to moderate to severe reticulin fibrosis (2? to 3?) (reticulin,oil immersion, original magnification ?500). C, Low-affinity nerve growth factor receptor staining. Adventitial reticular cells with delicate cyto-plasmic processes are highlighted. Some nonspecific staining is seen in plasma cells (immunohistochemistry, oil immersion, original magnification?500). D, Actin staining. Large vessels were highlighted by actin staining, with no actin staining seen in adjacent marrow elements. The presenceof actin staining would suggest myofibroblastic differentiation of the marrow stromal elements (immunohistochemistry, oil immersion, originalmagnification ?500). E, Collagen IV staining. Large vessels were outlined by collagen IV staining, highlighting basement membrane with someweak staining seen in adjacent sinusoids. No staining was seen in marrow interstitial spaces (immunohistochemistry, oil immersion, originalmagnification ?500).D). Collagen IV demonstrated weak staining of vesselwalls in only 5 of 35 cases of HIV/AIDS marrow biopsies(Figure, E). Linear staining was noted only in large-calibervessel walls. Focal staining of small-caliber vessels wasseen only rarely.Correlation of Hematologic and Histologic ParametersStatistical analysis did not reveal any correlation be-tween CD4 count and the degree of reticulin fibrosis.However, lower levels of LNGFR expression were associ-ated with CD4 counts of 100 or more (P ? .04 in univariateand multiple regression). In analyses of continuous CD4count, decreased LNGFR was associated with increasedCD4 count (P ? .03 and .02 in univariate and multipleregression, respectively).COMMENTA wide variety of hematologic abnormalities occur inHIV/AIDS patients, and many of these arise as a resultof direct and indirect effects of the disease on the bonemarrow. Stromal changes in the bone marrow and theirrelationship to hematologic parameters have not beenclosely examined in previous studies of HIV/AIDS-af-fected bone marrows.We found that marrow fibrosis, as measured by reticulinstaining, was present in all cases studied. As in other stud-ies, we found a statistically significant association betweenthe staining of ARCs and the degree of reticulinfibrosis.11–13No statistical correlation between CD4?lym-phocyte count and degree of reticulin fibrosis was iden-tified. This appears to suggest that the mechanism of mar-row fibrosis is independent of disease status, as measuredby CD4?lymphocyte counts.No significant expression of collagen IV was seen in theHIV/AIDS-affected bone marrows, except staining nor-mally seen in the walls of large vessels. Also, no signifi-cant actin staining was seen in the bone marrows studied.This suggests that the development of a ‘‘myofibroblastic’’phenotype is not a common pathway of stromal responsein HIV/AIDS-affected bone marrow.Megakaryocytes, ARCs, and monocyte/macrophagesmay all play a role in the development of stromal respons-es in bone marrow. With such diverse components con-

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[Show abstract][Hide abstract]ABSTRACT:
Systemic mastocytosis is a stem cell disorder characterized histologically by the presence of multifocal compact aggregates of mast cells in at least one extracutaneous organ with or without evidence of skin lesions. The mast cell aggregates are accompanied by fibrosis, which is often significant. However, in spite of its frequent occurrence and severity, little is known about its characteristics. In this study, we evaluated the composition of the fibrotic mast cell aggregates by studying eight bone marrow biopsies and two spleens involved by systemic mastocytosis, and compared the findings with those observed in other fibrotic bone marrow disorders such as primary myelofibrosis and metastatic malignancy. Histochemistry and immunohistochemistry were used to evaluate: (a) extracellular matrix (reticulin, trichrome, collagen IV, laminin); (b) stromal reticulum cells (low-affinity nerve growth factor receptor); (c) presence of myofibroblastic differentiation (smooth muscle actin) and (d) microvessel density (CD34). We found that all cases showed marked reticulin and collagen fibrosis. However, unlike primary myelofibrosis and metastatic malignancy, which are usually associated with increased low-affinity nerve growth factor receptor positivity, its expression was low in all cases of systemic mastocytosis. Myofibroblastic differentiation was only focally detected in two of eight bone marrow biopsies. In all cases, the systemic mastocytosis lesions were largely devoid of type IV collagen and laminin. The latter findings were in contrast with those seen in cases of primary myelofibrosis and metastatic malignancy where smooth muscle actin, collagen IV and laminin were expressed in most cases. Also in contrast with the other two conditions, only minimal vascularity was detectable within the fibrotic mast cell lesions. These findings indicate that systemic mastocytosis exhibits a distinct pattern of stromal change, and suggest that the fibrogenetic mechanism in systemic mastocytosis is most likely different from that of other bone marrow neoplasms which are also associated with fibrosis.

[Show abstract][Hide abstract]ABSTRACT:
To assess the prevalence, imaging appearance, and clinical significance, of bone marrow MR signal changes in a group of human immunodeficiency virus (HIV)-infected patients with lipodystrophy syndrome.
Twenty-eight HIV-infected patients with lipodystrophy syndrome treated with highly active antiretroviral therapy, and 12 HIV-negative controls underwent MRI of the legs. Whole-body MRI, SPECT/CT, and a complete radiographic skeletal survey were obtained in subjects with signal changes in bone marrow. MRI and clinical evaluations were reviewed 6 months after baseline to determine changes after switching from thymidine analogs (TA) to tenofovir-DF (TDF). MRI results correlated with clinical parameters.
We observed foci of a serous-like pattern (low signal and no enhancement on T1-weighted, high signal on T2-weighted images) in 4 out of 28 patients (14.3%) and an intermediate signal on T1-weighted images in 4 out of 28 patients (14.3%). Serous-like lesions were located in the lower limbs and scattered in the talus, calcaneus, femurs, and humeral bones; they showed slight uptake on SPECT bone scans and were normal on CT and radiographs. Patients with serous-like lesions had significantly lower peripheral and total fat at baseline than other groups (P < 0.05). No changes at 6 months were observed on MRI, and the serous-like lesion group showed good peripheral fat recovery after changing drug treatment.
A serous-like MRI pattern is observed in the peripheral skeletons of HIV-infected patients with lipodystrophy, which correlates with peripheral lipoatrophy, and should not be misdiagnosed as malignant or infectious diseases. Although the MR lesions did not improve after switching the treatment, there was evidence of lipoatrophy recovery.